Trivalent, bispecific antibodies

ABSTRACT

The present invention relates to trivalent, bispecific antibodies, methods for their production, pharmaceutical compositions containing the antibodies, and uses thereof.

PRIORITY TO RELATED APPLICATION(S)

This application is a continuation of U.S. Ser. No. 12/752,216, filedApr. 1, 2010, and claims the benefit of European Patent Application No.09005108.7, filed Apr. 7, 2009, which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 1, 2010 is named 26063.txt,and is 93,010 bytes in size.

FIELD OF THE INVENTION

The present invention relates to trivalent, bispecific antibodies,methods for their production, pharmaceutical compositions containing theantibodies, and uses thereof.

BACKGROUND OF THE INVENTION

A wide variety of multispecific recombinant antibody formats have beendeveloped in the recent past, e.g. tetravalent bispecific antibodies byfusion of, e.g., an IgG antibody format and single chain domains (seee.g. Coloma, M. J., et al., Nature Biotech 15 (1997) 159-163; WO2001/077342; and Morrison, S. L., Nature Biotech 25 (2007) 1233-1234).

Also several other new formats wherein the antibody core structure (IgA,IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- ortetrabodies, minibodies, several single chain formats (scFv, Bis-scFv),which are capable of binding two or more antigens, have been developed(Holliger, P., et al, Nature Biotech 23 (2005) 1126-1136; Fischer, N.,and Léger, O., Pathobiology 74 (2007) 3-14; Shen, J., et al., Journal ofImmunological Methods 318 (2007) 65-74; Wu, C., et al., Nature Biotech.25 (2007) 1290-1297).

All such formats use linkers either to fuse the antibody core (IgA, IgD,IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fusee.g. two Fab fragments or scFvs (Fischer, N., and Léger, O.,Pathobiology 74 (2007) 3-14). It has to be kept in mind that one maywant to retain effector functions, such as e.g. complement-dependentcytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC),which are mediated through the Fc receptor binding, by maintaining ahigh degree of similarity to naturally occurring antibodies.

In WO 2007/024715 are reported dual variable domain immunoglobulins asengineered multivalent and multispecific binding proteins. A process forthe preparation of biologically active antibody dimers is reported inU.S. Pat. No. 6,897,044. Multivalent F_(V) antibody construct having atleast four variable domains which are linked with each over via peptidelinkers are reported in U.S. Pat. No. 7,129,330. Dimeric and multimericantigen binding structures are reported in US 2005/0079170. Tri- ortetra-valent monospecific antigen-binding protein comprising three orfour Fab fragments bound to each other covalently by a connectingstructure, which protein is not a natural immunoglobulin are reported inU.S. Pat. No. 6,511,663. In WO 2006/020258 tetravalent bispecificantibodies are reported that can be efficiently expressed in prokaryoticand eukaryotic cells, and are useful in therapeutic and diagnosticmethods. A method of separating or preferentially synthesizing dimerswhich are linked via at least one interchain disulfide linkage fromdimers which are not linked via at least one interchain disulfidelinkage from a mixture comprising the two types of polypeptide dimers isreported in US 2005/0163782. Bispecific tetravalent receptors arereported in U.S. Pat. No. 5,959,083. Engineered antibodies with three ormore functional antigen binding sites are reported in WO 2001/077342.

Multispecific and multivalent antigen-binding polypeptides are reportedin WO 1997/001580. WO 1992/004053 reports homoconjugates, typicallyprepared from monoclonal antibodies of the IgG class which bind to thesame antigenic determinant are covalently linked by syntheticcross-linking. Oligomeric monoclonal antibodies with high avidity forantigen are reported in WO 1991/06305 whereby the oligomers, typicallyof the IgG class, are secreted having two or more immunoglobulinmonomers associated together to form tetravalent or hexavalent IgGmolecules. Sheep-derived antibodies and engineered antibody constructsare reported in U.S. Pat. No. 6,350,860, which can be used to treatdiseases wherein interferon gamma activity is pathogenic. In US2005/0100543 are reported targetable constructs that are multivalentcarriers of bi-specific antibodies, i.e., each molecule of a targetableconstruct can serve as a carrier of two or more bi-specific antibodies.Genetically engineered bispecific tetravalent antibodies are reported inWO 1995/009917. In WO 2007/109254 stabilized binding molecules thatconsist of or comprise a stabilized scFv are reported.

SUMMARY OF THE INVENTION

A first aspect of the current invention is a trivalent, bispecificantibody comprising

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains;

b) a polypeptide consisting of

ba) an antibody heavy chain variable domain (VH); or

bb) an antibody heavy chain variable domain (VH) and an antibodyconstant domain 1 (CH1), wherein the polypeptide is fused with theN-terminus of the VH domain via a peptide connector to the C-terminus ofone of the two heavy chains of the full length antibodyc) a polypeptide consisting ofca) an antibody light chain variable domain (VL), orcb) an antibody light chain variable domain (VL) and an antibody lightchain constant domain (CL);wherein the polypeptide is fused with the N-terminus of the VL domainvia a peptide connector to the C-terminus of the other of the two heavychains of the full length antibody;and wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) together form an antigen-binding sitespecifically binding to a second antigen

A further aspect of the invention is a nucleic acid molecule encoding atrivalent, bispecific antibody according to the invention.

Still further aspects of the invention are a pharmaceutical compositioncomprising the trivalent, bispecific antibody.

The trivalent, bispecific antibodies according to the invention one theone hand show new properties due to their binding to different antigens,and on the other hand are suitable for production and pharmaceuticalformulation due to their stability, low aggregation and pharmacokineticand biological properties. Due to their Ig core they still retain theproperties of natural antibodies like ADCC and CDC.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention is trivalent, bispecific antibody comprising

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains;

b) a polypeptide consisting of

ba) an antibody heavy chain variable domain (VH); or

bb) an antibody heavy chain variable domain (VH) and an antibodyconstant domain 1 (CH1), wherein the polypeptide is fused with theN-terminus of the VH domain via a peptide connector to the C-terminus ofone of the two heavy chains of the full length antibodyc) a polypeptide consisting ofca) an antibody light chain variable domain (VL), orcb) an antibody light chain variable domain (VL) and an antibody lightchain constant domain (CL);wherein the polypeptide is fused with the N-terminus of the VL domainvia a peptide connector to the C-terminus of the other of the two heavychains of the full length antibody;and wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) together form an antigen-binding sitespecifically binding to a second antigen

Optionally the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via a interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions:

i) heavy chain variable domain position 44 to light chain variabledomain position 100,

ii) heavy chain variable domain position 105 to light chain variabledomain position 43, or

iii) heavy chain variable domain position 101 to light chain variabledomain position 100 (numbering always according to EU index of Kabat).

Techniques to introduce unnatural disulfide bridges for stabilizationare described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Engin.(1997) 1453-59; Kobayashi, H., et al., Nuclear Medicine & Biology, Vol.25, (1998) 387-393; or Schmidt, M., et al., Oncogene (1999) 181711-1721. In one embodiment the optional disulfide bond between thevariable domains of the polypeptides under b) and c) is between heavychain variable domain position 44 and light chain variable domainposition 100. In one embodiment the optional disulfide bond between thevariable domains of the polypeptides under b) and c) is between heavychain variable domain position 105 and light chain variable domainposition 43. (numbering always according to EU index of Kabat) In oneembodiment a trivalent, bispecific antibody without the optionaldisulfide stabilization between the variable domains VH and VL of thesingle chain Fab fragments is preferred.

The term “full length antibody” denotes an antibody consisting of two“full length antibody heavy chains” and two “full length antibody lightchains” (see FIG. 1). A “full length antibody heavy chain” is apolypeptide consisting in N-terminal to C-terminal direction of anantibody heavy chain variable domain (VH), an antibody constant heavychain domain 1 (CH1), an antibody hinge region (HR), an antibody heavychain constant domain 2 (CH2), and an antibody heavy chain constantdomain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally anantibody heavy chain constant domain 4 (CH4) in case of an antibody ofthe subclass IgE. Preferably the “full length antibody heavy chain” is apolypeptide consisting in N-terminal to C-terminal direction of VH, CH1,HR, CH2 and CH3. A “full length antibody light chain” is a polypeptideconsisting in N-terminal to C-terminal direction of an antibody lightchain variable domain (VL), and an antibody light chain constant domain(CL), abbreviated as VL-CL. The antibody light chain constant domain(CL) can be κ (kappa) or λ (lambda). The two full length antibody chainsare linked together via inter-polypeptide disulfide bonds between the CLdomain and the CH1 domain and between the hinge regions of the fulllength antibody heavy chains. Examples of typical full length antibodiesare natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD,and IgE.) The full length antibodies according to the invention can befrom a single species e.g. human, or they can be chimerized or humanizedantibodies. The full length antibodies according to the inventioncomprise two antigen binding sites each formed by a pair of VH and VL,which both specifically bind to the same antigen. The C-terminus of theheavy or light chain of the full length antibody denotes the last aminoacid at the C-terminus of the heavy or light chain.

The N-terminus of the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) denotes the last amino acid at theN-terminus of VH or VL domain.

The CH3 domains of the full length antibody according to the inventioncan be altered by the “knob-into-holes” technology which is described indetail with several examples in e.g. WO 96/027011, Ridgway, J. B., etal., Protein Eng 9 (1996) 617-621; and Merchant, A. M., et al., NatBiotechnol 16 (1998) 677-681. In this method the interaction surfaces ofthe two CH3 domains are altered to increase the heterodimerisation ofboth heavy chains containing these two CH3 domains. Each of the two CH3domains (of the two heavy chains) can be the “knob”, while the other isthe “hole”. The introduction of a disulfide bridge further stabilizesthe heterodimers (Merchant, A. M., et al., Nature Biotech 16 (1998)677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) andincreases the yield.

Thus in one aspect of the invention the trivalent, bispecific antibodyis further is characterized in that the CH3 domain of one heavy chain ofthe full length antibody and the CH3 domain of the other heavy chain ofthe full length antibody each meet at an interface which comprises anoriginal interface between the antibody CH3 domains;

wherein the interface is altered to promote the formation of thebivalent, bispecific antibody, wherein the alteration is characterizedin that:

a) the CH3 domain of one heavy chain is altered,

so that within the original interface the CH3 domain of one heavy chainthat meets the original interface of the CH3 domain of the other heavychain within the bivalent, bispecific antibody, an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance within the interface of the CH3 domainof one heavy chain which is positionable in a cavity within theinterface of the CH3 domain of the other heavy chain andb) the CH3 domain of the other heavy chain is altered,so that within the original interface of the second CH3 domain thatmeets the original interface of the first CH3 domain within thetrivalent, bispecific antibodyan amino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within theinterface of the second CH3 domain within which a protuberance withinthe interface of the first CH3 domain is positionable.

Preferably the amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), tryptophan (W).

Preferably the amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), valine (V).

In one aspect of the invention both CH3 domains are further altered bythe introduction of cysteine (C) as amino acid in the correspondingpositions of each CH3 domain such that a disulfide bridge between bothCH3 domains can be formed.

In a preferred embodiment, the trivalent, bispecific comprises a T366Wmutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407Vmutations in the CH3 domain of the “hole chain”. An additionalinterchain disulfide bridge between the CH3 domains can also be used(Merchant, A. M., et al., Nature Biotech 16 (1998) 677-681) e.g. byintroducing a Y349C mutation into the CH3 domain of the “knobs chain”and a E356C mutation or a S354C mutation into the CH3 domain of the“hole chain”. Thus in a another preferred embodiment, the trivalent,bispecific antibody comprises Y349C, T366W mutations in one of the twoCH3 domains and E356C, T366S, L368A, Y407V mutations in the other of thetwo CH3 domains or the trivalent, bispecific antibody comprises Y349C,T366W mutations in one of the two CH3 domains and S354C, T366S, L368A,Y407V mutations in the other of the two CH3 domains (the additionalY349C mutation in one CH3 domain and the additional E356C or S354Cmutation in the other CH3 domain forming a interchain disulfide bridge)(numbering always according to EU index of Kabat). But also otherknobs-in-holes technologies as described by EP 1 870 459A1, can be usedalternatively or additionally. A preferred example for the trivalent,bispecific antibody are R409D; K370E mutations in the CH3 domain of the“knobs chain” and D399K; E357K mutations in the CH3 domain of the “holechain” (numbering always according to EU index of Kabat).

In another preferred embodiment the trivalent, bispecific antibodycomprises a T366W mutation in the CH3 domain of the “knobs chain” andT366S, L368A, Y407V mutations in the CH3 domain of the “hole chain” andadditionally R409D; K370E mutations in the CH3 domain of the “knobschain” and D399K; E357K mutations in the CH3 domain of the “hole chain”.

In another preferred embodiment the trivalent, bispecific antibodycomprises Y349C, T366W mutations in one of the two CH3 domains andS354C, T366S, L368A, Y407V mutations in the other of the two CH3 domainsor the trivalent, bispecific antibody comprises Y349C, T366W mutationsin one of the two CH3 domains and S354C, T366S, L368A, Y407V mutationsin the other of the two CH3 domains and additionally R409D; K370Emutations in the CH3 domain of the “knobs chain” and D399K; E357Kmutations in the CH3 domain of the “hole chain”.

The bispecific antibody to the invention comprises three antigen-bindingsites (A) the full length antibody according comprises two identicalantigen-binding sites specifically binding to a first antigen, and B)the antibody heavy chain variable domain (VH) of the polypeptide underb) and the antibody light chain variable domain (VL) of the polypeptideunder c) form together one antigen binding site specifically binding toa second antigen). The terms “binding site” or “antigen-binding site” asused herein denotes the region(s) of the bispecific antibody accordingto the invention to which the respective antigen actually specificallybinds. The antigen binding sites either in the full length antibody orby the antibody heavy chain variable domain (VH) of the polypeptideunder b) and the antibody light chain variable domain (VL) of thepolypeptide under c) are formed each by a pair consisting of an antibodylight chain variable domain (VL) and an antibody heavy chain variabledomain (VH).

The antigen-binding sites that specifically bind to the desired antigencan be derived a) from known antibodies to the antigen or b) from newantibodies or antibody fragments obtained by de novo immunizationmethods using inter alia either the antigen protein or nucleic acid orfragments thereof or by phage display.

An antigen-binding site of an antibody of the invention contains sixcomplementarity determining regions (CDRs) which contribute in varyingdegrees to the affinity of the binding site for antigen. There are threeheavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and threelight chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent ofCDR and framework regions (FRs) is determined by comparison to acompiled database of amino acid sequences in which those regions havebeen defined according to variability among the sequences.

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific. “Bispecific antibodies” according to the invention areantibodies which have two different antigen-binding specificities. Wherean antibody has more than one specificity, the recognized epitopes maybe associated with a single antigen or with more than one antigen. Theterm “monospecific” antibody as used herein denotes an antibody that hasone or more binding sites each of which bind to the same epitope of thesame antigen. The term “valent” as used within the current applicationdenotes the presence of a specified number of binding sites in anantibody molecule. A natural antibody for example or a full lengthantibody according to the invention has two binding sites and isbivalent. As such, the terms “trivalent”, denote the presence of threebinding sites in an antibody molecule. The bispecific antibodiesaccording to the invention are “trivalent”. The term “trivalent,bispecific” antibody as used herein denotes an antibody that has threeantigen-binding sites of which two bind to the same antigen (or the sameepitope of the antigen) and the third binds to a different antigen or adifferent epitope of the same antigen. Antibodies of the presentinvention have three binding sites and are bispecific.

Another embodiment of the current invention is a trivalent, bispecificantibody comprising:

a) a full length antibody specifically binding to a first antigen andconsisting of:

aa) two antibody heavy chains consisting in N-terminal to C-terminaldirection of an antibody heavy chain variable domain (VH), an antibodyconstant heavy chain domain 1 (CH1), an antibody hinge region (HR), anantibody heavy chain constant domain 2 (CH2), and an antibody heavychain constant domain 3 (CH3); andab) two antibody light chains consisting in N-terminal to C-terminaldirection of an antibody light chain variable domain (VL), and anantibody light chain constant domain (CL); andb) a polypeptide consisting ofba) an antibody heavy chain variable domain (VH); orbb) an antibody heavy chain variable domain (VH) and an antibodyconstant domain 1 (CH1), wherein the polypeptide is fused with theN-terminus of the VH domain via a peptide connector to the C-terminus ofone of the two heavy chains of the full length antibody wherein thepeptide connector is a peptide of at least 5 amino acids, preferablybetween 25 and 50 amino acids;c) a polypeptide consisting ofca) an antibody light chain variable domain (VL), orcb) an antibody light chain variable domain (VL) and an antibody lightchain constant domain (CL);wherein the polypeptide is fused with the N-terminus of the VL domainvia a peptide connector to the C-terminus of the other of the two heavychains of the full length antibody;wherein the peptide connector is identical to the peptide connectorunder b);and wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) together form an antigen-binding sitespecifically binding to a second antigen.

Within this embodiment, preferably the trivalent, bispecific antibodycomprises a T366W mutation in one of the two CH3 domains of and T366S,L368A, Y407V mutations in the other of the two CH3 domains and morepreferably the trivalent, bispecific antibody comprises Y349C, T366Wmutations in one of the two CH3 domains of and D356C, T366S, L368A,Y407V mutations in the other of the two CH3 domains (the additionalY349C mutation in one CH3 domain and the additional D356C mutation inthe other CH3 domain forming a interchain disulfide bridge).

In one embodiment of the invention the trivalent, bispecific antibodyaccording to the invention is characterized in that

a) the full length antibody is specifically binding to ErbB-3 comprisesas heavy chain variable domain the sequence of SEQ ID NO: 1, and aslight chain variable domain the sequence of SEQ ID NO: 2

b) the polypeptide under b) comprises as the heavy chain variable domainthe sequence of SEQ ID NO: 3; and

c) the polypeptide under c) comprises as the light chain variable domainthe sequence of SEQ ID NO: 4.

In another aspect of the current invention the trivalent, bispecificantibody according to the invention comprises

a) a full length antibody binding to a first antigen consisting of twoantibody heavy chains VH-CH1-HR-CH2-CH3 and two antibody light chainsVL-CL;

(wherein preferably one of the two CH3 domains comprises Y349C, T366Wmutations and the other of the two CH3 domains comprises S354C, T366S,L368A, Y407V mutations);

b) a polypeptide consisting of

ba) an antibody heavy chain variable domain (VH); or

bb) an antibody heavy chain variable domain (VH) and an antibodyconstant domain 1 (CH1), wherein the polypeptide is fused with theN-terminus of the VH domain via a peptide connector to the C-terminus ofone of the two heavy chains of the full length antibodyc) a polypeptide consisting ofca) an antibody light chain variable domain (VL), orcb) an antibody light chain variable domain (VL) and an antibody lightchain constant domain (CL);wherein the polypeptide is fused with the N-terminus of the VL domainvia a peptide connector to the C-terminus of the other of the two heavychains of the full length antibody;and wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) together form an antigen-binding sitespecifically binding to a second antigen.

Another embodiment of the current invention is a trivalent, bispecificantibody comprising

a) a full length antibody specifically binding to human ErbB-3 andconsisting of:

aa) two antibody heavy chains consisting in N-terminal to C-terminaldirection of an antibody heavy chain variable domain (VH), an antibodyconstant heavy chain domain 1 (CH1), an antibody hinge region (HR), anantibody heavy chain constant domain 2 (CH2), and an antibody heavychain constant domain 3 (CH3); andab) two antibody light chains consisting in N-terminal to C-terminaldirection of an antibody light chain variable domain (VL), and anantibody light chain constant domain (CL) (VL-CL); andb) one single chain Fv fragment specifically binding to human c-Met),wherein the single chain Fv fragment under b) is fused to the fulllength antibody under a) via a peptide connector at the C- or N-terminusof the heavy or light chain (preferably at the C-terminus of the heavychain) of the full length antibody;wherein the peptide connector is a peptide of at least 5 amino acids,preferably between 25 and 50 amino acids.

Preferably such trivalent, bispecific antibody further comprises Y349C,T366W mutations in one of the two CH3 domains of the full lengthantibody and S354C (or E356C), T366S, L368A, Y407V mutations in theother of the two CH3 domains of the full length antibody.

Another embodiment of the current invention is a trivalent, bispecificantibody comprising

a) a full length antibody specifically binding to human ErbB-3 andconsisting of:

aa) two antibody heavy chains consisting in N-terminal to C-terminaldirection of an antibody heavy chain variable domain (VH), an antibodyconstant heavy chain domain 1 (CH1), an antibody hinge region (HR), anantibody heavy chain constant domain 2 (CH2), and an antibody heavychain constant domain 3 (CH3); andab) two antibody light chains consisting in N-terminal to C-terminaldirection of an antibody light chain variable domain (VL), and anantibody light chain constant domain (CL); andb) a polypeptide consisting ofba) an antibody heavy chain variable domain (VH); orbb) an antibody heavy chain variable domain (VH) and an antibodyconstant domain 1 (CH1), wherein the polypeptide is fused with theN-terminus of the VH domain via a peptide connector to the C-terminus ofone of the two heavy chains of the full length antibody wherein thepeptide connector is a peptide of at least 5 amino acids, preferablybetween 25 and 50 amino acids;c) a polypeptide consisting ofca) an antibody light chain variable domain (VL), orcb) an antibody light chain variable domain (VL) and an antibody lightchain constant domain (CL);wherein the polypeptide is fused with the N-terminus of the VL domainvia a peptide connector to the C-terminus of the other of the two heavychains of the full length antibody;wherein the peptide connector is identical to the peptide connectorunder b);and wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) together form an antigen-binding sitespecifically binding to human c-Met

The full length antibodies of the invention comprise immunoglobulinconstant regions of one or more immunoglobulin classes. Immunoglobulinclasses include IgG, IgM, IgA, IgD, and IgE isotypes and, in the case ofIgG and IgA, their subtypes. In a preferred embodiment, a full lengthantibody of the invention has a constant domain structure of an IgG typeantibody.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to an antibody comprising a variableregion, i.e., binding region, from one source or species and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a murine variable region and a human constant region arepreferred. Other preferred forms of “chimeric antibodies” encompassed bythe present invention are those in which the constant region has beenmodified or changed from that of the original antibody to generate theproperties according to the invention, especially in regard to C1qbinding and/or Fc receptor (FcR) binding. Such chimeric antibodies arealso referred to as “class-switched antibodies.”. Chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding immunoglobulin variable regions and DNA segmentsencoding immunoglobulin constant regions. Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques are well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No.5,202,238 and U.S. Pat. No. 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric antibodies. Other forms of “humanizedantibodies” encompassed by the present invention are those in which theconstant region has been additionally modified or changed from that ofthe original antibody to generate the properties according to theinvention, especially in regard to C1q binding and/or Fc receptor (FcR)binding.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well-known in thestate of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin.Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced intransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire or a selection of human antibodies in theabsence of endogenous immunoglobulin production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge(see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;Brüggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodiescan also be produced in phage display libraries (Hoogenboom, H. R., andWinter, G. J., Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J.Mol. Biol. 222 (1991) 581-597). The techniques of Cole, S. P. C., etal., and Boerner, P., et al., are also available for the preparation ofhuman monoclonal antibodies (Cole, S. P. C., et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77-96 (1985); andBoerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentionedfor chimeric and humanized antibodies according to the invention theterm “human antibody” as used herein also comprises such antibodieswhich are modified in the constant region to generate the propertiesaccording to the invention, especially in regard to C1q binding and/orFcR binding, e.g. by “class switching” i.e. change or mutation of Fcparts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutation.)

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions in arearranged form. The recombinant human antibodies according to theinvention have been subjected to in vivo somatic hypermutation. Thus,the amino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangerm line VH and VL sequences, may not naturally exist within the humanantibody germ line repertoire in vivo.

The “variable domain” (variable domain of a light chain (VL), variableregion of a heavy chain (VH) as used herein denotes each of the pair oflight and heavy chains which is involved directly in binding theantibody to the antigen. The domains of variable human light and heavychains have the same general structure and each domain comprises fourframework (FR) regions whose sequences are widely conserved, connectedby three “hypervariable regions” (or complementarity determiningregions, CDRs). The framework regions adopt a β-sheet conformation andthe CDRs may form loops connecting the β-sheet structure. The CDRs ineach chain are held in their three-dimensional structure by theframework regions and form together with the CDRs from the other chainthe antigen binding site. The antibody heavy and light chain CDR3regions play a particularly important role in the bindingspecificity/affinity of the antibodies according to the invention andtherefore provide a further object of the invention.

The terms “hypervariable region” or “antigen-binding portion of anantibody” when used herein refer to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from the “complementaritydetermining regions” or “CDRs”. “Framework” or “FR” regions are thosevariable domain regions other than the hypervariable region residues asherein defined. Therefore, the light and heavy chains of an antibodycomprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. CDRs on each chain are separated by such framework aminoacids. Especially, CDR3 of the heavy chain is the region whichcontributes most to antigen binding. CDR and FR regions are determinedaccording to the standard definition of Kabat, E. A., et al., Sequencesof Proteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the antigen in an in vitroassay, preferably in an plasmon resonance assay (BIAcore, GE-HealthcareUppsala, Sweden) with purified wild-type antigen. The affinity of thebinding is defined by the terms ka (rate constant for the association ofthe antibody from the antibody/antigen complex), k_(D) (dissociationconstant), and K_(D) (k_(D)/ka). Binding or specifically binding means abinding affinity (K_(D)) of 10⁻⁸ mol/l or less, preferably 10⁻⁹ M to10⁻¹³ mol/l. Thus, an trivalent, bispecific antibody according to theinvention is specifically binding to each antigen for which it isspecific with a binding affinity (K_(D)) of 10⁻⁸ mol/l or less,preferably 10⁻⁹ M to 10⁻¹³ mol/l.

Binding of the antibody to the FcγRIII can be investigated by a BIAcoreassay (GE-Healthcare Uppsala, Sweden). The affinity of the binding isdefined by the terms ka (rate constant for the association of theantibody from the antibody/antigen complex), k_(D) (dissociationconstant), and K_(D) (k_(D)/ka).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody.

In certain embodiments, an antibody is the to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

The term “peptide connector” as used within the invention denotes apeptide with amino acid sequences, which is preferably of syntheticorigin. These peptide connectors according to invention are used to fusethe polypeptides under b) and c) to the heavy chain C-termini of thefull length antibody to form the trivalent, bispecific antibodyaccording to the invention. Preferably the peptide connectors arepeptides with an amino acid sequence with a length of at least 5 aminoacids, preferably with a length of 10 to 100 amino acids, morepreferably with a length of 25 to 50 amino acids. Preferably the peptideconnector under b) and c) are identical peptides with a length of atleast 25 amino acids, preferably with a length between 25 and 50 aminoacids and more preferably the peptide connector is (G×S)n or (G×S)nGmwith G=glycine, S=serine, and (x=3, n=6, 7 or 8, and m=0, 1, 2 or 3) or(x=4, n=3, 4, 5, 6, or 7 and m=0, 1, 2 or 3), preferably x=4 and n=5, 6,or 7.

In a further embodiment the trivalent, bispecific antibody according tothe invention is characterized in that the full length antibody is ofhuman IgG1 subclass, or of human IgG1 subclass with the mutations L234Aand L235A.

In a further embodiment the trivalent, bispecific antibody according tothe invention is characterized in that the full length antibody is ofhuman IgG2 subclass.

In a further embodiment the trivalent, bispecific antibody according tothe invention is characterized in that the full length antibody is ofhuman IgG3 subclass.

In a further embodiment the trivalent, bispecific antibody according tothe invention is characterized in that the full length antibody is ofhuman IgG4 subclass or, of human IgG4 subclass with the additionalmutation S228P.

Preferably the trivalent, bispecific antibody according to the inventionis characterized in that the full length antibody is of human IgG1subclass, of human IgG4 subclass with the additional mutation S228P.

It has now been found that the trivalent, bispecific antibodiesaccording to the invention have improved characteristics such asbiological or pharmacological activity, pharmacokinetic properties ortoxicity. They can be used e.g. for the treatment of diseases such ascancer.

In a further embodiment the trivalent, bispecific antibody according tothe invention is characterized in specifically binding to ErbB3 andc-Met. The term “constant region” as used within the currentapplications denotes the sum of the domains of an antibody other thanthe variable region. The constant region is not involved directly inbinding of an antigen, but exhibit various effector functions. Dependingon the amino acid sequence of the constant region of their heavy chains,antibodies are divided in the classes: IgA, IgD, IgE, IgG and IgM, andseveral of these may be further divided into subclasses, such as IgG1,IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain constant regionsthat correspond to the different classes of antibodies are called α, δ,ε, γ, and μ, respectively. The light chain constant regions (CL) whichcan be found in all five antibody classes are called κ (kappa) and λ(lambda).

The term “constant region derived from human origin” as used in thecurrent application denotes a constant heavy chain region of a humanantibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constantlight chain kappa or lambda region. Such constant regions are well knownin the state of the art and e.g. described by Kabat, E. A., (see e.g.Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat,E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).

While antibodies of the IgG4 subclass show reduced Fc receptor(FcγRIIIa) binding, antibodies of other IgG subclasses show strongbinding. However Pro238, Asp265, Asp270, Asn297 (loss of Fccarbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254,Lys288, Thr307, Gln311, Asn434, and His435 are residues which, ifaltered, provide also reduced Fc receptor binding (Shields, R. L., etal., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al., FASEB J. 9(1995) 115-119; Morgan, A., et al., Immunology 86 (1995) 319-324; EP 0307 434).

In one embodiment an antibody according to the invention has a reducedFcR binding compared to an IgG1 antibody and the full length parentantibody is in regard to FcR binding of IgG4 subclass or of IgG1 or IgG2subclass with a mutation in S228, L234, L235 and/or D265, and/orcontains the PVA236 mutation. In one embodiment the mutations in thefull length parent antibody are S228P, L234A, L235A, L235E and/orPVA236. In another embodiment the mutations in the full length parentantibody are in IgG4 S228P and in IgG1 L234A and L235A.

The constant region of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity). Complement activation (CDC) isinitiated by binding of complement factor C1q to the constant region ofmost IgG antibody subclasses. Binding of C1q to an antibody is caused bydefined protein-protein interactions at the so called binding site. Suchconstant region binding sites are known in the state of the art anddescribed e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981)2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979)907-917; Burton, D. R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E. E., et al.,J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75(2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324;and EP 0 307 434. Such constant region binding sites are, e.g.,characterized by the amino acids L234, L235, D270, N297, E318, K320,K322, P331, and P329 (numbering according to EU index of Kabat).

The term “antibody-dependent cellular cytotoxicity (ADCC)” refers tolysis of human target cells by an antibody according to the invention inthe presence of effector cells. ADCC is measured preferably by thetreatment of a preparation of antigen expressing cells with an antibodyaccording to the invention in the presence of effector cells such asfreshly isolated PBMC or purified effector cells from buffy coats, likemonocytes or natural killer (NK) cells or a permanently growing NK cellline.

The term “complement-dependent cytotoxicity (CDC)” denotes a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3.

Cell-mediated effector functions of monoclonal antibodies can beenhanced by engineering their oligosaccharide component as described inUmana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and U.S. Pat.No. 6,602,684. IgG1 type antibodies, the most commonly used therapeuticantibodies, are glycoproteins that have a conserved N-linkedglycosylation site at Asn297 in each CH2 domain. The two complexbiantennary oligosaccharides attached to Asn297 are buried between theCH2 domains, forming extensive contacts with the polypeptide backbone,and their presence is essential for the antibody to mediate effectorfunctions such as antibody dependent cellular cytotoxicity (ADCC)(Lifely, M. R., et al., Glycobiology 5 (1995) 813-822; Jefferis, R., etal., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, S. L.,Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al. NatureBiotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpressionin Chinese hamster ovary (CHO) cells ofβ(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofantibodies. Alterations in the composition of the Asn297 carbohydrate orits elimination affect also binding to FcγR and C1q (Umana, P., et al.,Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al., Biotechnol.Bioeng. 74 (2001) 288-294; Mimura, Y., et al., J. Biol. Chem. 276 (2001)45539-45547; Radaev, S., et al., J. Biol. Chem. 276 (2001) 16478-16483;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R.L., et al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L. C., etal., J. Immunol. Methods 263 (2002) 133-147).

Methods to enhance cell-mediated effector functions of monoclonalantibodies are reported e.g. in WO 2005/018572, WO 2006/116260, WO2006/114700, WO 2004/065540, WO 2005/011735, WO 2005/027966, WO1997/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO2003/035835, WO 2000/061739.

Surprisingly the bispecific <ErbB3-c-Met> antibodies which are oneembodiment of the invention show reduced downregulation andinternalization of target antigen compared to their parent <ErbB3>and/or <c-Met> antibodies. Therefore in one preferred embodiment of theinvention, the bispecific antibody is glycosylated (if it comprises anFc part of IgG1, IgG2, IgG3 or IgG4 subclass, preferably of IgG1 or IgG3subclass) with a sugar chain at Asn297 whereby the amount of fucosewithin the sugar chain is 65% or lower (Numbering according to Kabat).In another embodiment is the amount of fucose within the sugar chain isbetween 5% and 65%, preferably between 20% and 40%. “Asn297” accordingto the invention means amino acid asparagine located at about position297 in the Fc region. Based on minor sequence variations of antibodies,Asn297 can also be located some amino acids (usually not more than ±3amino acids) upstream or downstream of position 297, i.e. betweenposition 294 and 300. In one embodiment the glycosylated antibodyaccording to the invention the IgG subclass is of human IgG1 subclass,of human IgG1 subclass with the mutations L234A and L235A or of IgG3subclass. In a further embodiment the amount of N-glycolylneuraminicacid (NGNA) is 1% or less and/or the amount of N-terminalalpha-1,3-galactose is 1% or less within the sugar chain. The sugarchain show preferably the characteristics of N-linked glycans attachedto Asn297 of an antibody recombinantly expressed in a CHO cell.

The term “the sugar chains show characteristics of N-linked glycansattached to Asn297 of an antibody recombinantly expressed in a CHO cell”denotes that the sugar chain at Asn297 of the full length parentantibody according to the invention has the same structure and sugarresidue sequence except for the fucose residue as those of the sameantibody expressed in unmodified CHO cells, e.g. as those reported in WO2006/103100.

The term “NGNA” as used within this application denotes the sugarresidue N-glycolylneuraminic acid.

Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylatedbiantennary complex oligosaccharide glycosylation terminated with up totwo Gal residues. Human constant heavy chain regions of the IgG1 or IgG3subclass are reported in detail by Kabat, E. A., et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), and by Brüggemann,M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T. W., et al.,Methods Enzymol. 178 (1989) 515-527. These structures are designated asG0, G1 (α-1,6- or α-1,3-), or G2 glycan residues, depending from theamount of terminal Gal residues (Raju, T. S., Bioprocess Int. 1 (2003)44-53). CHO type glycosylation of antibody Fc parts is e.g. described byRoutier, F. H., Glycoconjugate J. 14 (1997) 201-207. Antibodies whichare recombinantly expressed in non-glycomodified CHO host cells usuallyare fucosylated at Asn297 in an amount of at least 85%. The modifiedoligosaccharides of the full length parent antibody may be hybrid orcomplex. Preferably the bisected, reduced/not-fucosylatedoligosaccharides are hybrid. In another embodiment, the bisected,reduced/not-fucosylated oligosaccharides are complex.

According to the invention “amount of fucose” means the amount of thesugar within the sugar chain at Asn297, related to the sum of allglycostructures attached to Asn297 (e.g. complex, hybrid and highmannose structures) measured by MALDI-TOF mass spectrometry andcalculated as average value. The relative amount of fucose is thepercentage of fucose-containing structures related to allglycostructures identified in an N-Glycosidase F treated sample (e.g.complex, hybrid and oligo- and high-mannose structures, resp.) byMALDI-TOF.

The antibody according to the invention is produced by recombinantmeans. Thus, one aspect of the current invention is a nucleic acidencoding the antibody according to the invention and a further aspect isa cell comprising the nucleic acid encoding an antibody according to theinvention. Methods for recombinant production are widely known in thestate of the art and comprise protein expression in prokaryotic andeukaryotic cells with subsequent isolation of the antibody and usuallypurification to a pharmaceutically acceptable purity. For the expressionof the antibodies as aforementioned in a host cell, nucleic acidsencoding the respective modified light and heavy chains are insertedinto expression vectors by standard methods. Expression is performed inappropriate prokaryotic or eukaryotic host cells like CHO cells, NS0cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant orcells after lysis). General methods for recombinant production ofantibodies are well-known in the state of the art and described, forexample, in the review articles of Makrides, S. C., Protein Expr. Purif.17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996)271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-160; Werner, R.G., Drug Res. 48 (1998) 870-880.

The trivalent, bispecific antibodies according to the invention aresuitably separated from the culture medium by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography. DNA and RNA encoding themonoclonal antibodies is readily isolated and sequenced usingconventional procedures. The hybridoma cells can serve as a source ofsuch DNA and RNA. Once isolated, the DNA may be inserted into expressionvectors, which are then transfected into host cells such as HEK 293cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of the trivalent, bispecificantibody are prepared by introducing appropriate nucleotide changes intothe antibody DNA, or by nucleotide synthesis. Such modifications can beperformed, however, only in a very limited range, e.g. as describedabove. For example, the modifications do not alter the above mentionedantibody characteristics such as the IgG isotype and antigen binding,but may improve the yield of the recombinant production, proteinstability or facilitate the purification.

The term “host cell” as used in the current application denotes any kindof cellular system which can be engineered to generate the antibodiesaccording to the current invention. In one embodiment HEK293 cells andCHO cells are used as host cells. As used herein, the expressions“cell,” “cell line,” and “cell culture” are used interchangeably and allsuch designations include progeny. Thus, the words “transformants” and“transformed cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Variant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included.

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

A nucleic acid is “operably linked” when it is placed in a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Purification of antibodies is performed in order to eliminate cellularcomponents or other contaminants, e.g. other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis, and otherswell known in the art. See Ausubel, F., et al., ed. Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York(1987). Different methods are well established and widespread used forprotein purification, such as affinity chromatography with microbialproteins (e.g. protein A or protein G affinity chromatography), ionexchange chromatography (e.g. cation exchange (carboxymethyl resins),anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilicadsorption (e.g. with beta-mercaptoethanol and other SH ligands),hydrophobic interaction or aromatic adsorption chromatography (e.g. withphenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid),metal chelate affinity chromatography (e.g. with Ni(II)- andCu(II)-affinity material), size exclusion chromatography, andelectrophoretical methods (such as gel electrophoresis, capillaryelectrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75(1998) 93-102).

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

One embodiment of the invention is the trivalent, bispecific antibodyaccording to the invention for the treatment of cancer.

Another aspect of the invention is the pharmaceutical composition forthe treatment of cancer.

Another aspect of the invention is the use of an antibody according tothe invention for the manufacture of a medicament for the treatment ofcancer.

Another aspect of the invention is method of treatment of patientsuffering from cancer by administering an antibody according to theinvention to a patient in the need of such treatment.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term cancer as used herein refers to proliferative diseases, such aslymphomas, lymphocytic leukemias, lung cancer, non small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F. L., and van der Eb, A. J., Virology 52 (1973) 456-467.However, other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S. N, et al., PNAS 69 (1972) 2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

Description of the Amino Acid Sequences

SEQ ID NO:1 heavy chain variable domain <ErbB3> HER3 clone 29

SEQ ID NO:2 light chain variable domain <ErbB3> HER3 clone 29

SEQ ID NO:3 heavy chain variable domain <c-Met> Mab 5D5

SEQ ID NO:4 light chain variable domain <c-Met> Mab 5D5

SEQ ID NO:5 heavy chain <ErbB3> HER3 clone 29

SEQ ID NO:6 light chain <ErbB3> HER3 clone 29

SEQ ID NO:7 heavy chain <c-Met> Mab 5D5

SEQ ID NO:8 light chain <c-Met> Mab 5D5

SEQ ID NO:9 heavy chain <c-Met> Fab 5D5

SEQ ID NO:10 light chain <c-Met> Fab 5D5

SEQ ID NO:11 heavy chain 1 <ErbB3-c-Met> Her3/Met_KHSS

SEQ ID NO:12 heavy chain 2 <ErbB3-c-Met> Her3/Met_KHSS

SEQ ID NO:13 light chain <ErbB3-c-Met> Her3/Met_KHSS

SEQ ID NO:14 heavy chain 1 <ErbB3-c-Met> Her3/Met_SSKH

SEQ ID NO:15 heavy chain 2 <ErbB3-c-Met> Her3/Met_SSKH

SEQ ID NO:16 light chain <ErbB3-c-Met> Her3/Met_SSKH

SEQ ID NO:17 heavy chain 1 <ErbB3-c-Met> Her3/Met_SSKHSS

SEQ ID NO:18 heavy chain 2 <ErbB3-c-Met> Her3/Met_SSKHSS

SEQ ID NO:19 light chain <ErbB3-c-Met> Her3/Met_SSKHSS

SEQ ID NO:20 heavy chain 1 <ErbB3-c-Met> Her3/Met_1C

SEQ ID NO:21 heavy chain 2 <ErbB3-c-Met> Her3/Met_1C

SEQ ID NO:22 light chain <ErbB3-c-Met> Her3/Met_1C

SEQ ID NO:23 heavy chain 1 <ErbB3-c-Met> Her3/Met_6C

SEQ ID NO:24 heavy chain 2 <ErbB3-c-Met> Her3/Met_6C

SEQ ID NO:25 light chain <ErbB3-c-Met> Her3/Met_6C

SEQ ID NO:26 heavy chain constant region of human IgG1

SEQ ID NO:27 heavy chain constant region of human IgG1

SEQ ID NO:28 human light chain kappa constant region

SEQ ID NO:29 human light chain lambda constant region

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic structure of a full length antibody without CH4 domainspecifically binding to a first antigen 1 with two pairs of heavy andlight chain which comprise variable and constant domains in a typicalorder.

FIG. 2 Schematic representation of a trivalent, bispecific antibodyaccording to the invention, comprising a full length antibodyspecifically binding to a first antigen 1 to which

a) FIG. 2a two polypeptides VH and VL are fused (the VH and VL domainsof both together forming a antigen binding site specifically binding toa second antigen 2;

b) FIG. 2b two polypeptides VH-CH1 and VL-CL are fused (the VH and VLdomains of both together forming a antigen binding site specificallybinding to a second antigen 2)

FIG. 3 Schematic representation of a trivalent, bispecific antibodyaccording to the invention, comprising a full length antibodyspecifically binding to a first antigen 1 to which two polypeptides VHand VL are fused (the VH and VL domains of both together forming aantigen binding site specifically binding to a second antigen 2) with“knobs and holes”.

FIG. 4 Schematic representation of a trivalent, bispecific antibodyaccording to the invention, comprising a full length antibodyspecifically binding to a first antigen 1 to which two polypeptides VHand VL are fused (the VH and VL domains of both together forming aantigen binding site specifically binding to a second antigen 2, whereinthese VH and VL domains comprise an interchain disulfide bridge betweenpositions VH44 and VL100) with “knobs and holes”.

FIG. 5 Binding of bispecific antibodies to the cell surface of cancercells

FIG. 6 Inhibition of HGF-induced c-Met receptor phosphorylation bybispecific Her3/c-Met antibody formats in different cell lines. FIG. 6a) A549 cells, FIG. 6b ) HT29 cells, and FIG. 6c ) HT29 cells.

FIG. 7 Inhibition of HRG-induced Her3 receptor phosphorylation bybispecific Her3/c-Met antibody formats. FIG. 7a ) Phosphorylation ofHer3 in MCF7 cells, and FIG. 7b ) Inhibition of PhosphoHER3 at 0.1 μg/mland 1 μg/ml.

FIG. 8 Inhibition of HGF-induced HUVEC proliferation by bispecificHer3/c-Met antibody formats

FIG. 9 Inhibition of proliferation in the cancer cell line A431 bybispecific Her3/c-Met antibody formats.

FIG. 10 Analysis of inhibition of HGF-induced cell-cell dissemination(scattering) in the cancer cell line A431 by bispecific Her3/c-Metantibody formats.

EXPERIMENTAL PROCEDURE Examples Materials & Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

DNA and Protein Sequence Analysis and Sequence Data Management

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A. et al.,(1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIHPublication No 91-3242. Amino acids of antibody chains are numberedaccording to EU numbering (Edelman, G. M., et al., PNAS 63 (1969) 78-85;Kabat, E. A., et al., (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Ed., NIH Publication No 91-3242). The GCG's (GeneticsComputer Group, Madison, Wis.) software package version 10.2 andInfomax's Vector NTI Advance suite version 8.0 was used for sequencecreation, mapping, analysis, annotation and illustration.DNA Sequencing

DNA sequences were determined by double strand sequencing performed atSequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).

Gene Synthesis

Desired gene segments were prepared by Geneart AG (Regensburg, Germany)from synthetic oligonucleotides and PCR products by automated genesynthesis. The gene segments which are flanked by singular restrictionendonuclease cleavage sites were cloned into pGA18 (ampR) plasmids. Theplasmid DNA was purified from transformed bacteria and concentrationdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene Segments coding“knobs-into-hole” Her3 (clone 29) antibody heavy chain carrying a T366Wmutation in the CH3 domain with a C-terminal 5D5 VH region linked by a(G₄S)_(n) peptide connector as well as “knobs-into-hole” Her3 (clone 29)antibody heavy chain carrying T366S, L368A and Y407V mutations with aC-terminal 5D5 VL region linked by a (G₄S)_(n) peptide connector weresynthesized with 5′-BamHI and 3′-XbaI restriction sites. In a similarmanner, DNA sequences coding “knobs-into-hole” Her3 (clone 29) antibodyheavy chain carrying S354C and T366W mutations in the CH3 domain with aC-terminal 5D5 VH region linked by a (G₄S)_(n) peptide connector as wellas “knobs-into-hole” Her3 (clone 29) antibody heavy chain carryingY349C, T366S, L368A and Y407V mutations with a C-terminal 5D5 VL regionlinked by a (G₄S)_(n) peptide connector were prepared by gene synthesiswith flanking BamHI and XbaI restriction sites. Finally, DNA sequencesencoding unmodified heavy and light chains of the Her3 (clone 29) and5D5 antibody were synthesized with flanking BamHI and XbaI restrictionsites. All constructs were designed with a 5′-end DNA sequence codingfor a leader peptide (MGWSCIILFLVATATGVHS), which targets proteins forsecretion in eukaryotic cells.

Construction of the Expression Plasmids

A Roche expression vector was used for the construction of all heavyVH/or VL fusion protein and light chain protein encoding expressionplasmids. The vector is composed of the following elements:

-   -   a hygromycin resistance gene as a selection marker,    -   an origin of replication, oriP, of Epstein-Barr virus (EBV),    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli,    -   the immediate early enhancer and promoter from the human        cytomegalovirus (HCMV),    -   the human 1-immunoglobulin polyadenylation (“poly A”) signal        sequence, and    -   unique BamHI and XbaI restriction sites.

The immunoglobulin fusion genes comprising the heavy or light chainconstructs as well as “knobs-into-hole” constructs with C-terminal VHand VL domains were prepared by gene synthesis and cloned into pGA18(ampR) plasmids as described. The pG18 (ampR) plasmids carrying thesynthesized DNA segments and the Roche expression vector were digestedwith BamHI and XbaI restriction enzymes (Roche Molecular Biochemicals)and subjected to agarose gel electrophoresis. Purified heavy and lightchain coding DNA segments were then ligated to the isolated Rocheexpression vector BamHI/XbaI fragment resulting in the final expressionvectors. The final expression vectors were transformed into E. colicells, expression plasmid DNA was isolated (Miniprep) and subjected torestriction enzyme analysis and DNA sequencing. Correct clones weregrown in 150 ml LB-Amp medium, again plasmid DNA was isolated (Maxiprep)and sequence integrity confirmed by DNA sequencing.

Transient Expression of Immunoglobulin Variants in HEK293 Cells

Recombinant immunoglobulin variants were expressed by transienttransfection of human embryonic kidney 293-F cells using the FreeStyle™293 Expression System according to the manufacturer's instruction(Invitrogen, USA). Briefly, suspension FreeStyle™ 293-F cells werecultivated in FreeStyle™ 293 Expression medium at 37° C./8% CO₂ and thecells were seeded in fresh medium at a density of 1-2×10⁶ viablecells/ml on the day of transfection. DNA-293Fectin™ complexes wereprepared in Opti-MEM® I medium (Invitrogen, USA) using 325 μl of293Fectin™ (Invitrogen, Germany) and 250 μg of heavy and light chainplasmid DNA in a 1:1 molar ratio for a 250 ml final transfection volume.“Knobs-into-hole” DNA-293fectin complexes were prepared in Opti-MEM® Imedium (Invitrogen, USA) using 325 μl of 293Fectin™ (Invitrogen,Germany) and 250 μg of “Knobs-into-hole” heavy chain 1 and 2 and lightchain plasmid DNA in a 1:1:2 molar ratio for a 250 ml final transfectionvolume. Antibody containing cell culture supernatants were harvested 7days after transfection by centrifugation at 14000 g for 30 minutes andfiltered through a sterile filter (0.22 μm). Supernatants were stored at−20° C. until purification.

Purification of Trivalent Bispecific and Control Antibodies

Trivalent bispecific and control antibodies were purified from cellculture supernatants by affinity chromatography using ProteinA-Sepharose™ (GE Healthcare, Sweden) and Superdex200 size exclusionchromatography. Briefly, sterile filtered cell culture supernatants wereapplied on a HiTrap ProteinA HP (5 ml) column equilibrated with PBSbuffer (10 mM Na₂HPO₄, 1 mM KH₂PO₄, 137 mM NaCl and 2.7 mM KCl, pH 7.4).Unbound proteins were washed out with equilibration buffer. Antibody andantibody variants were eluted with 0.1 M citrate buffer, pH 2.8, and theprotein containing fractions were neutralized with 0.1 ml 1 M Tris, pH8.5. Then, the eluted protein fractions were pooled, concentrated withan Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) to avolume of 3 ml and loaded on a Superdex200 HiLoad 120 ml 16/60 gelfiltration column (GE Healthcare, Sweden) equilibrated with 20 mMHistidin, 140 mM NaCl, pH 6.0. Fractions containing purified bispecificand control antibodies with less than 5% high molecular weightaggregates were pooled and stored as 1.0 mg/ml aliquots at −80° C. Fabfragments were generated by a Papain digest of the purified 5D5monoclonal antibody and subsequent removal of contaminating Fc domainsby Protein A chromatography. Unbound Fab fragments were further purifiedon a Superdex200 HiLoad 120 ml 16/60 gel filtration column (GEHealthcare, Sweden) equilibrated with 20 mM Histidin, 140 mM NaCl, pH6.0, pooled and stored as 1.0 mg/ml aliquots at −80° C.

Analysis of Purified Proteins

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of bispecific and control antibodies were analyzedby SDS-PAGE in the presence and absence of a reducing agent (5 mM1,4-dithiotreitol) and staining with Coomassie brilliant blue). TheNuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to themanufacturer's instruction (4-20% Tris-Glycine gels). The aggregatecontent of bispecific and control antibody samples was analyzed byhigh-performance SEC using a Superdex 200 analytical size-exclusioncolumn (GE Healthcare, Sweden) in 200 mM KH₂PO₄, 250 mM KCl, pH 7.0running buffer at 25° C. 25 μg protein were injected on the column at aflow rate of 0.5 ml/min and eluted isocratic over 50 minutes. Forstability analysis, concentrations of 1 mg/ml of purified proteins wereincubated at 4° C. and 40° C. for 7 days and then evaluated byhigh-performance SEC. The integrity of the amino acid backbone ofreduced bispecific antibody light and heavy chains was verified byNanoElectrospray Q-TOF mass spectrometry after removal of N-glycans byenzymatic treatment with Peptide-N-Glycosidase F (Roche MolecularBiochemicals).

c-Met Phosphorylation Assay

5×10e5 A549 cells were seeded per well of a 6-well plate the day priorHGF stimulation in RPMI with 0.5% FCS (fetal calf serum). The next day,growth medium was replaced for one hour with RPMI containing 0.2% BSA(bovine serum albumine). 5 μg/mL of the bispecific antibody was thenadded to the medium and cells were incubated for 10 minutes upon whichHGF was added for further 10 minutes in a final concentration of 50ng/mL. Cells were washed once with ice cold PBS containing 1 mM sodiumvanadate upon which they were placed on ice and lysed in the cellculture plate with 100 μL lysis buffer (50 mM Tris-Cl pH7.5, 150 mMNaCl, 1% NP40, 0.5% DOC, aprotinine, 0.5 mM PMSF, 1 mM sodium-vanadate).Cell lysates were transferred to eppendorf tubes and lysis was allowedto proceed for 30 minutes on ice. Protein concentration was determinedusing the BCA method (Pierce). 30-50 μg of the lysate was separated on a4-12% Bis-Tris NuPage gel (Invitrogen) and proteins on the gel weretransferred to a nitrocellulose membrane. Membranes were blocked for onehour with TBS-T containing 5% BSA and developed with a phospho-specificc-Met antibody directed against Y1230, 1234, 1235 (44-888, Biosource)according to the manufacturer's instructions. Immunoblots were reprobedwith an antibody binding to unphosphorylated c-Met (AF276, R&D).

Her3 (ErbB3) Phosphorylation Assay

2×10e5 MCF7 cells were seeded per well of a 12-well plate in completegrowth medium (RPMI 1640, 10% FCS). Cells were allowed to grow to 90%confluency within two days. Medium was then replaced with starvationmedium containing 0.5% FCS. The next day the respective antibodies weresupplemented at the indicated concentrations 1 hour prior addition of500 ng/mL Heregulin (R&D). Upon addition of Heregulin cells werecultivated further 10 minutes before the cells were harvested and lysed.Protein concentration was determined using the BCA method (Pierce).30-50 μg of the lysate was separated on a 4-12% Bis-Tris NuPage gel(Invitrogen) and proteins on the gel were transferred to anitrocellulose membrane. Membranes were blocked for one hour with TBS-Tcontaining 5% BSA and developed with a phospho-specific Her3/ErbB3antibody specifically recognizing Tyr1289 (4791, Cell Signaling).

Scatter Assay

A549 (4000 cells per well) or A431 (8000 cells per well) were seeded theday prior compound treatment in a total volume of 200 μL in 96-wellE-Plates (Roche, 05232368001) in RPMI with 0.5% FCS. Adhesion and cellgrowth was monitored over night with the Real Time Cell Analyzer machinewith sweeps every 15 min monitoring the impedance. The next day, cellswere pre-incubated with 5 μL of the respective antibody dilutions in PBSwith sweeps every five minutes. After 30 minutes 2.5 μL of a HGFsolution yielding a final concentration of 20 ng/mL were added and theexperiment was allowed to proceed for further 72 hours. Immediatechanges were monitored with sweeps every minute for 180 minutes followedby sweeps every 15 minutes for the remainder of the time.

FACS

a) Binding Assay

A431 were detached and counted. 1.5×10e5 cells were seeded per well of aconical 96-well plate. Cells were spun down (1500 rpm, 4° C., 5 min) andincubated for 30 min on ice in 50 μL of a dilution series of therespective bispecific antibody in PBS with 2% FCS (fetal calf serum).Cells were again spun down and washed once with 200 μL PBS containing 2%FCS followed by a second incubation of 30 min with aphycoerythrin-coupled antibody directed against human Fc which wasdiluted in PBS containing 2% FCS (Jackson Immunoresearch, 109116098).Cells were spun down washed twice with 200 μL PBS containing 2% FCS,resuspended in BD CellFix solution (BD Biosciences) and incubated for atleast 10 min on ice. Mean fluorescence intensity (mfi) of the cells wasdetermined by flow cytometry (FACS Canto, BD). Mfi was determined atleast in duplicates of two independent stainings Flow cytometry spectrawere further processed using the FlowJo software (TreeStar).Half-maximal binding was determined using XLFit 4.0 (IDBS) and the doseresponse one site model 205.

b) Internalization Assay

Cells were detached and counted. 5×10e5 cells were placed in 50 μLcomplete medium in an eppendorf tube and incubated with 5 μg/mL of therespective bispecific antibody at 37° C. After the indicated time pointscells were stored on ice until the time course was completed.Afterwards, cells were transferred to FACS tubes, spun down (1500 rpm,4° C., 5 min), washed with PBS+2% FCS and incubated for 30 minutes in 50μL phycoerythrin-coupled secondary antibody directed against human Fcwhich was diluted in PBS containing 2% FCS (Jackson Immunoresearch,109116098). Cells were again spun down, washed with PBS+2% FCS andfluorescence intensity was determined by flow cytometry (FACS Canto,BD).

c) Crosslinking Experiment

HT29 cells were detached counted and split in two populations which wereindividually stained with PKH26 and PKH67 (Sigma) according to themanufacturer's instructions. Of each of the stained populations 5×10e5cells were taken, combined and incubated for 30 and 60 minutes with 10μg/mL of the respective bispecific antibody in complete medium. Afterthe indicated time points cells were stored on ice until the time coursewas completed. Cells were spun down (1500 rpm, 4° C., 5 min), washedwith PBS+2% FCS and fluorescence intensity was determined by flowcytometry (FACS Canto, BD).

Cell Titer Glow Assay

Cell viability and proliferation was quantified using the cell titerglow assay (Promega). The assay was performed according to themanufacturer's instructions. Briefly, cells were cultured in 96-wellplates in a total volume of 100 μL for the desired period of time. Forthe proliferation assay, cells were removed from the incubator andplaced at room temperature for 30 min. 100 μL of cell titer glow reagentwere added and multi-well plates were placed on an orbital shaker for 2min. Luminescence was quantified after 15 min on a microplate reader(Tecan).

Wst-1 Assay

A Wst-1 viability and cell proliferation assay was performed as endpointanalysis, detecting the number of metabolic active cells. Briefly, 20 μLof Wst-1 reagent (Roche, 11644807001) were added to 200 μL of culturemedium. 96-well plates were further incubated for 30 min to 1 h untilrobust development of the dye. Staining intensity was quantified on amicroplate reader (Tecan) at a wavelength of 450 nm.

Design of Expressed and Purified Trivalent, Bispecific <ErbB3-c-Met>Antibodies

In Table 1: Trivalent, bispecific <ErbB3-c-Met> antibodies based on afull length ErbB-3 antibody (HER3 clone29) and the VH and VL domain froma C-met antibody (c-Met 5D5) with the respective features shown inTable1 one were expressed and purified according to the general methodsdescribed above. The corresponding VH and VL of HER3 clone29 and c-Met5D5 are given in the sequence listing.

TABLE 1 Trivalent, bispecific antibody < ErbB3-c-Met> with theVHVL-Ab-nomenclature in Table 1 were expressed and purified (see also inthe Examples below and FIG. 3c) Molecule Name VHVL-Ab-nomenclature forbispecific antibodies Features: Her3/Met_KHSS Her3/Met_SSKHHer3/Met_SSKHSS Her3/Met_1C Her3/Met_6C Knobs-in-hole S354C:T366W/T366W/ S354C:T366W/ S354C:T366W/ S354C:T366W/ mutations Y349′C:T366′S:T366′S:L368′A: Y349′C:T366′S: Y349′C:T366′S: Y349′C:T366′S:L368′A:Y407′V Y407′V L368′A:Y407′V L368′A:Y407′V L368′A:Y407′V Fulllength Her3 Her3 Her3 Her3 Her3 antibody clone 29 clone 29 clone 29clone 29 clone 29 backbone (chimeric) (chimeric) (chimeric) (chimeric)(chimeric) derived from VHVLfragment cMet 5D5 cMet 5D5 cMet 5D5 cMet 5D5cMet 5D5 derived from (humanized) (humanized) (humanized) (humanized)(humanized) Position of VH C-terminus C-terminus C-terminus C-terminusC-terminus attached to knob heavy knob heavy knob heavy knob heavy knobheavy antibody chain chain chain chain chain Position of VL C-terminusC-terminus C-terminus C-terminus C-terminus attached to hole heavy holeheavy hole heavy hole heavy hole heavy antibody chain chain chain chainchain Peptide (G₄S)₃ (G₄S)₃ (G₄S)₃ (G₄S)₁ (G₄S)₆ connector VHVLdisulfide − + + − − VH44/VL100 stabilized

Example 1 (FIG. 5) Binding of Bispecific Antibodies to the Cell Surfaceof Cancer Cells

The binding properties of the bispecific antibodies to their respectivereceptor on the cell surface was analyzed on A431 cancer cells in a flowcytometry based assay. Cells were incubated with the mono- or bispecificprimary antibodies and binding of these antibodies to their cognatereceptors was detected with a secondary antibody coupled to afluorophore binding specifically to the Fc of the primary antibody. Themean fluorescence intensity of a dilution series of the primaryantibodies was plotted against the concentration of the antibody toobtain a sigmoidal binding curve. Cell surface expression of c-Met andHer3 was validated by incubation with the bivalent 5D5 and Her3 clone 29antibody only. The Her3/c-Met_KHSS antibody readily binds to the cellsurface of A431. Under these experimental settings, the antibody canonly bind via its Her3 part and consequently the mean fluorescenceintensity does not exceed the staining for Her3 clone 29 alone.

Example 2 (FIG. 6) Inhibition of HGF-Induced c-Met ReceptorPhosphorylation by Bispecific Her3/c-Met Antibody Formats

To confirm functionality of the c-Met part in the bispecific antibodiesa c-Met phosphorylation assay was performed. In this experiment A549lung cancer cells or HT29 colorectal cancer cells were treated with thebispecific antibodies or control antibodies prior exposure to HGF. Cellswere then lysed and phosphorylation of the c-Met receptor was examined.Both cell lines can be stimulated with HGF as can be observed by theoccurrence of a phospho-c-Met specific band in the immunoblot.

Example 3 (FIG. 7) Inhibition of HRG-Induced Her3 ReceptorPhosphorylation by Bispecific Her3/c-Met Antibody Formats

To confirm functionality of the Her3 part in the bispecific antibodies aHer3 phosphorylation assay was performed. In this experiment MCF7 cellswere treated with the bispecific antibodies or control antibodies priorexposure to HRG (Heregulin). Cells were then lysed and phosphorylationof the Her3 receptor was examined. Her3/c-Met_KHSS inhibit Her3 receptorphosphorylation to the same extent as the parental Her3 clone29indicating that Her3 binding and functionality of the antibody are notcompromised by the trivalent antibody format.

Example 4 (FIG. 8) Inhibition of HGF-Induced HUVEC Proliferation byBispecific Her3/c-Met Antibody Formats

HUVEC proliferation assays were performed to demonstrate the mitogeniceffect of HGF. Addition of HGF to HUVEC leads to a twofold increase inproliferation. Addition of human IgG control antibody in the sameconcentration range as the bispecific antibodies has no impact oncellular proliferation while the 5D5 Fab fragment inhibits HGF-inducedproliferation. Titration of Her3/c-Met_KHSS demonstrate a weakinhibitory effect of the antibody (FIG. 8). The effect is morepronounced for the Her3/Met-6C antibody indicating that a longerconnector improves efficacy of the antibody. This demonstrates thefunctionality of the c-Met component in the trivalent antibody format.

Example 5 (FIG. 9) Inhibition of Proliferation in the Cancer Cell LineA431 by Bispecific Her3/c-Met Antibody Formats

If A431 were seeded in serum reduced medium, addition of HGF inducesapart from scattering a weak mitogenic effect. This was exploited toanalyze the impact of Her3/c-Met_KHSS on HGF treated A431 proliferation.Indeed, the bispecific antibodies can largely inhibit the HGF-inducedincrease of proliferation (15%). A control human IgG1 antibody has noinfluence on HGF promoted A431 cell growth.

Example 6 (FIG. 10) Analysis of Inhibition of HGF-Induced Cell-CellDissemination (Scattering) in the Cancer Cell Line A431 by BispecificHer3/c-Met Antibody Formats

HGF-induced scattering includes morphological changes of the cell,resulting in rounding of the cells, filopodia-like protrusions,spindle-like structures and a certain motility of the cells. The RealTime Cell Analyzer (Roche) measures the impedance of a given cellculture well and can therefore indirectly monitor changes in cellularmorphology and proliferation. Addition of HGF to A431 and A549 cellsresulted in changes of the impedance which was monitored as function oftime. Her3/c-Met_KHSS and Her3/Met-6C inhibited HGF-induced scatteringwith Her3/Met-6C being more efficacious (20.7% and 43.7% scatterinhibition) (FIG. 10).

What is claimed is:
 1. A trivalent, bispecific antibody comprising a) afull length IgG1 antibody that specifically binds to a first antigenwherein the full length antibody consists of two antibody heavy chainsand two antibody light chains, wherein the CH3 domain of one heavy chainand the CH3 domain of the other heavy chain each meet at an interfacewhich comprises an alteration in the original interface between theantibody CH3 domains, wherein i) in the CH3 domain of one heavy chain anamino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theinterface of the CH3 domain of one heavy chain which is positionable ina cavity within the interface of the CH3 domain of the other heavy chainand wherein ii) in the CH3 domain of the other heavy chain an amino acidresidue is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the interface of thesecond CH3 domain within which a protuberance within the interface ofthe first CH3 domain is positionable; b) a polypeptide consisting of ba)an antibody heavy chain variable domain (VH); or bb) an antibody heavychain variable domain (VH) and an antibody constant domain 1 (CH1),wherein the N-terminus of the VH domain of the polypeptide is fused viaa peptide connector to the C-terminus of one of the two heavy chains ofthe full length antibody; c) a polypeptide consisting of ca) an antibodylight chain variable domain (VL), or cb) an antibody light chainvariable domain (VL) and an antibody light chain constant domain (CL);wherein N-terminus of the VL domain of the polypeptide is fused via apeptide connector to the C-terminus of the other of the two heavy chainsof the full length antibody; wherein the antibody heavy chain variabledomain (VH) of the polypeptide under b) and the antibody light chainvariable domain (VL) of the polypeptide under c) together form anantigen-binding site specifically binding to a second antigen; andwherein the peptide connectors under b) and c) are peptides with alength between 5 and 50 amino acids.
 2. The trivalent, bispecificantibody according to claim 1, wherein i) the amino acid residue havinga larger side chain volume is selected from the group consisting ofarginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W); and ii)the amino acid residue having a smaller side chain volume is selectedfrom the group consisting of alanine (A), serine (S), threonine (T),valine (V).
 3. The trivalent, bispecific antibody according to claim 2,wherein both CH3 domains are further altered by the introduction ofcysteine as an amino acid in each CH3 domain such that a disulfidebridge between both CH3 domains can be formed.
 4. The trivalent,bispecific antibody according to claim 3, wherein the CH3 domain underi) comprises a T366W mutation; and the CH3 domain under ii) comprisesT366S, L368A, and Y407V mutations.
 5. The trivalent, bispecific antibodyaccording to claim 4, wherein the CH3 domain under i) comprises Y349Cand T366W mutations; and the CH3 domain under ii) comprises S354C,T366S, L368A, and Y407V mutations.
 6. The trivalent, bispecific antibodyaccording to claim 3, wherein the antibody heavy chain variable domain(VH) of the polypeptide under b) and the antibody light chain variabledomain (VL) of the polypeptide under c) are linked and stabilized via ainterchain disulfide bridge by introduction of a disulfide bond betweenthe following positions: i) heavy chain variable domain position 44 tolight chain variable domain position 100, ii) heavy chain variabledomain position 105 to light chain variable domain position 43, or iii)heavy chain variable domain position 101 to light chain variable domainposition
 100. 7. The trivalent, bispecific antibody according to claim6, wherein the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via a interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions: i) heavy chain variable domain position 44 to lightchain variable domain position
 100. 8. The trivalent, bispecificantibody according to claim 5, wherein the peptide connectors under b)and c) are identical peptides with a length between 25 and 50 aminoacids.
 9. A pharmaceutical composition comprising a trivalent,bispecific antibody according to claim
 1. 10. The trivalent, bispecificantibody according to claim 1, wherein the peptide connectors comprisesGlycine (G) and Serine (S) residues.
 11. The trivalent, bispecificantibody according to claim 10, wherein the peptide connector comprisesGGGGS (SEQ ID NO:34) repeats.